Imprinting

Question 1

What are epigenetics modifications? 1x example

Answer 1

Changes in gene expression that do not alter the DNA sequence. Stable in mitosis so are passed on in somatic cells. Critical for development and cell processes

DNA methylation

Question 2

How does DNA methylation regulate gene expression?

Answer 2

CH3 added to C5 of cytosine= 5 methylcytosine (5meC) usually present at CpG dinucleotides

Methyl groups are recognised by MeCpG binding proteins, recruit HDACs and other repressive proteins, close chromatin conformation = silences gene expression

Question 3

Name and function of DNA methyltransferases - 2x examples

Answer 3

DNMT1 = maintenance, copies meth pattern from partner strand during replication

DNMT3A/B = de novo, active in early embryo and add the initial methylation patterns

Question 4

What is imprinting?

Answer 4

Epigenetic parent of origin expression i.e only expressed from maternal or paternal allele (not biparentally)

Question 5

Define ICR and DMR

Answer 5

Imprinting Control Regions regulate clusters of imprinted genes

Differentially Methylated Regions are epigenetic modifications of ICRs that determine if a gene is expressed

Question 6

Why are imprinted genes/regions susceptible to disease?

Answer 6

Imprinted regions have a functional haploid state, meaning a single variant is required to deregulate function

Mostly locus specific but global methylation disorders have been described

Question 7

4 mechanisms of imprinting disorders

Answer 7

Chromosome rearrangement

SNV in the active allele

Epimutation e.g. hypo/hypermeth

UPD

Question 8

Imprinted chromosomes (n=6) and related disorders

Answer 8

UPD(6)pat = transient neonatal diabetes mellitus (PLAGL1)

UPD(7)mat = RSS

UPD(11)pat = BWS

UPD(14)mat = Temple syndrome (MEG3-DMR)

UPD(15)pat = AS
UPD(15)mat = PWS

UPD(20q)pat = Pseudohypoparathyroidism type 1b (GNAS)

Question 9

What is uniparental isodisomy and how does it arise?

Answer 9

2 identical copies of a homolog from same parent. Meiosis II or mitotic errors

Question 10

What is uniparental heterodisomy and how does it arise?

Answer 10

2 homologs from same parent. Meiosis I error

Question 11

Give an example of whole genome UPD

Answer 11

Complete hydatidiform mole - whole genome paternal UPD. Placenta overgrowth and no fetus.

90% = 46,XX, empty egg + 1 sperm that duplicates

10% = 46,XX or XY. empty egg + 2 sperm

Have 15% risk of becoming invasive and 3% risk of transforming to choriocarcinoma

Question 12

Describe 2 mechanisms of whole chromosome UPD

Answer 12

Trisomy rescue (post-zygotically) due to
- meiosis I NDJ (usually maternal). 2 homologs fail to separate > rescue of other parent homolog > UPHD
- meiosis II NDJ. Sister chromatids fail to separate > rescue of other parent homolog > UPID

Monosomy rescue due to meiosis I/II NDJ (usually maternal). Nullisomic gamate > fertilised > Haploid embryo > Dup of single homolog > UPID (usually mat)
Window of rescue is short as monosomy is lethal early in development

Question 13

How does segmental UPD occur?

Answer 13

Post-zygotic mitotic exchange between sister chromatids that were originally biparental. This results in somatic mosaicism of different UPDs

Question 14

1 example of aberrant methylation in cancer

Answer 14

MLH1 promoter hypermethylation = microsatellite instability and predicts sporadic CRC not hereditary

Question 15

1 example of epigenetic therapy

Answer 15

Azacytidine, for MDS, is a cytosine analog that incorporates into DNA, blocks DNMT, decreases methylation and reactivates silenced genes

Question 16

Describe the 15q11-q13 imprinted locus
(3 main components)

Answer 16

SNURF/SNRPN encodes a single long transcript of snoRNAs that are highly expressed in the brain. Expressed on paternal allele.

UBE3A encodes an E3 ubiquitin ligase that adds polyUb chains to target substrates for proteasome degradation. Essential function in neuronal synapses. Expressed on maternal allele.

ICR is a bipartite structure: PWS-ICR (paternal, unmethylated) and AS-ICR (maternal, methylated)

Question 17

PWS main mechanism and clinical features

Answer 17

Loss of paternal allele at 15q11q13 (hypermethylation)

Hypotonia, dev delay, hyperphagia, obesity, behavioural difficulties, OCD, hypogonadism, incomplete puberty

Question 18

AS main mechanism and clinical features

Answer 18

Loss of maternal allele at 15q11q13 (hypomethylation or UBE3A mut)

Severe dev delay, no language, gait ataxia, LD, epilepsy, microcephaly, hyperactivity, unique happy demenour

Question 19

PWS - x4 mechanisms

Answer 19

De novo deletion of paternal chr - 70-75% (RR<1% *)

UPD(15)mat - 25-30% (RR<1% *)

Imprinting defect - 1%
> 10-15% have an IC deletion (RR=50% if in father)

*Unless parent = translocation carrier

Question 20

AS - x6 mechanisms

Answer 20

De novo deletion of maternal chr -75% (RR<1% *)

UPD(15)pat - 1-2% (RR<1% *)

Imprinting defect - 3%
> 10-15% have an IC deletion (RR=50% if in mother)

UBE3A mutation - 5-10% (RR=50% if in mother)

No identifiable abn in 10-15% (RR up to 50%)

*Unless parent = translocation carrier

Question 21

What is the common 15q11-q13 deletion?

Answer 21

60% = BP2-BP3 ~5Mb

These have more severe phenotypes than UPD/imprinting defect

Question 22

Describe the 11p15 imprinted locus

Answer 22

2 ICRs

ICR1 / IG-DMR
- Unmeth + CTCF on maternal allele = H19 expression
- Meth on paternal allele = IGF2 expression (growth promoter). No CTCF allows IGF2 promoter/enhancer interaction. Loss of IGF2 = growth restriction in RSS

ICR2 / TSS-DMR
- Meth on maternal allele = KCNQ1 and CDKN1C expression
- Unmeth on paternal allele = KCNQ1OT1 expression

Question 23

BWS clinical features

Why is is difficult to test prenatally?

Answer 23

Overgrowth disorder with exomphalos, macroglossia, Wilms tumour, hyperinsulinism, high birth weight, polyhydramnios, placentomegaly, neuroblastoma

Methylation patterns may not be establish at time of CVS sampling and patterns may be different in placenta/fetus. Culturing may also affect methylation.

Question 24

BWS mechanisms (n=6)

Answer 24

ICR2 hypometh (loss of mat). 50-60%. RR low

ICR1 hypermath (gain of pat). 5-10%. RR low unless mat ICR1 microdel

UPD(11)pat. 20-25%. RR low unless translocation

CDKN1C mutation. 5% de novo, 50% familial. RR=50% depending on parent carrier

Pat DUP or Mat DEL. 1-2%. RR=50% depending on parent carrier

Mosaic paternal unidiploidy. 1%. RR=0 (de novo)

Question 25

RSS clinical features

Answer 25

Pre and postnatal growth retardation, short stature, relative macrocephaly at birth, protruding forehead, body asymmetry, triangular face

Question 26

RSS mechanisms (n=3)

+rare causes

Answer 26

ICR1 hypometh (loss of pat). 40-60%. RR=low

Mat DUP or Pat DEL. <1%. RR=50% depending on parent carrier

UPD(7)mat. 4-10%. RR low unless translocation

Rare causes = UPD(11)mat, CDKN1C activating maternal mutation, IGF2 LOF paternal mutation, mosaic maternal unidiploidy

Question 27

Basis of MS-MLPA

Answer 27

Denaturation, hybridisation, ligation, into 2 reactions:
> Undigested (CNVs)
> Digested with methylation sensitive RE Hhal

Unmethylated = digestion = no amplification
Methylated = no digestion = amplification

Question 28

Advantages & limitations of MS-MLPA, microsatellite analysis and SNP array for PWS/AS testing

Answer 28

MS-MLPA is a simple/robust assay to identify CNV and hypo/hypermeth but cannot distinguish UPD and IC defects

Microsatellite analysis confirms UPD or IC defect (if biparental) but requires parent bloods to compare STRs surrounding locus and only if enough informative markers

SNP arrays can detect CNVs, iso/hetero UPD and is a genome wide not targeted assay (increases number of informative markers). Parent samples are required to confirm true UPD (rather than IBD) and is expensive

Important to determine PWS/AS mechanism for accurate recurrence risks